To conduct a research study on aquatic microorganisms, including minute crustaceans, such as copepod, cycrops, daphnia, Gammarus, euphasiid, mysidopsis bahia, cumacea, and triopsidae spp.; large crustaceans, such as nauplius, cypris, zoea, and mince larvae; zooplanktons, such as rotifer and ameba; and phytoplanktons, such as cyanophyceae, chlorophyta, and symbiodinium sp., morphological observation under a microscope is absolutely indispensable. In terms of taxonomy, in particular, for the larvae of minute crustaceans and large crustaceans, importance is given to morphological observation of fine legs called appendages. To make a detailed observation of the appendages of crustaceans, which are complicatedly entangled, the appendages need to be separated from each other for anatomy. Conventionally, the appendages of the crustaceans have been separated from each other for anatomy under a light microscope; however, control of anatomical devices is difficult under the light microscope, requiring skilled technique for anatomy.
In contrast, an electronic microscope with deep depth of focus is suitable to the observation of solid forms of living organisms and probes capable of controlling samples in the sample chamber of the electronic microscope are available. When aquatic microorganisms are taken out from water, however, for exposure to the atmosphere, they are too dried and shrunk to the degree that their original forms are not retained. The larvae of Minute crustaceans and large crustaceans, in particular, are deformed due to drying and shrinkage even when they are fixed using any of fixatives such as ethanol, glutaraldehyde, and formalin. When the aquatic organisms are observed under the electronic microscope, it is required to take out them from water or encapsulating medium and take them into a vacuum environment; for this reason, it has been difficult to observe the samples without pre-processing under the electronic microscope.
To solve this problem, methods for pre-processing samples for electronic microscope have been developed. A typical example of these methods is a method that prevents rupture of morphology of soft biological tissues using a low-vacuum cryostage for electron microscope capable of controlling the temperature of samples in the range from several tens of degrees below zero to room temperature in the low-vacuum atmosphere (1.3 to 270 Pa). For instance, in a nonpatent literature 1, it is disclosed that cryo-electron microscopy has been successfully achieved on Ipomoea nil leaf primordium using the aforementioned method.
On the other hand, a method using ionic liquid has been developed as a method for observing and controlling minute samples in a liquid under an electronic microscope. In Patent Literature 1, it is disclosed that such a property of ionic liquid that it does not evaporate even in vacuum is used to enable biological samples to be observed in their original forms under the electronic microscope. Disclosed are the methods, in particular, characterized in that samples containing water are immersed in the ionic liquid and then water is removed under vacuum; and the ionic liquid diluted with any of solvents such as alcohol is coated onto samples and then the solvent is removed under vacuum.
Since the aforementioned invention, the development of the methods for observing biological samples using ionic liquid has been promoted. For instance, in the nonpatent literature 2, it is reported that when ionic liquid was dripped on the colonies of acid-fast bacteria cultivated in an agar medium, which had been vapor-fixed with osmic acid, images, which seemed to be the original structures of the colonies of acid-fast bacteria, were obtained.
The patent literature 2 discloses that a certain ionic liquid enhances permeability into permeability into biological samples compared with other ionic liquid. In embodiments, the observation examples of the stems of Undaria pinnatifida and Spinacia oleracea, cotton tissue, hair, mouse bone, erythrocyte, and Streptococcus mutans are exemplified. Moreover, a method for impregnating, applying, and spraying a fluid medium, which is prepared by dissolving the ionic liquid in a polar solvent such as water, on the samples is disclosed.